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• Jassowicz, Lena; Feng, Fangyoumin; Warta, Rolf; Heinicke, Fatima; Wöllner, Amélie & Briesch, Hannah [Show all 43 contributors for this article] (2026). Dissecting the cellular architecture of breast cancer brain metastases reveals prognostically distinct immune landscapes. Cancer Cell. ISSN 1535-6108. doi: 10.1016/j.ccell.2026.03.016. Full text in Research Archive
• Rydland, Anne; Heinicke, Fatima; Nyman, Tuula Anneli; Siebke, Anne-Marie; Flåm, Siri Tennebø & Stensland, Maria [Show all 11 contributors for this article] (2025). Newly-diagnosed rheumatoid arthritis patients have elevated levels of plasma extracellular vesicles with protein cargo altered towards inflammatory processes. Scientific Reports. 15(1). doi: 10.1038/s41598-025-96325-8. Full text in Research Archive Show summary

  Extracellular vesicles (EVs) are implicated in rheumatoid arthritis (RA) but have mainly been assessed in RA patients taking disease modifying anti-rheumatic drugs. EVs are nanoparticles important in cell-cell communication and their molecular cargo are biomarker candidates. We characterized the protein profiles of EVs from blood plasma from newly diagnosed, treatment naïve RA patients (N = 32) and compared them to healthy controls (N = 20), by size exclusion chromatography-based EV enrichment coupled with high-resolution quantitative proteomics. The RA patients had higher EV concentration and larger EVs than controls. A total of 682 EV proteins were reliably quantified, and the overall profiles were distinctly different between patients and controls. Specifically, 26 proteins were significantly upregulated and 31 downregulated in RA patients, with several proteins acting in inflammatory networks and with immunologically important upstream regulators. The RA associated EVs appear, based on the tissue expression of their cargo proteins, to originate mainly from hepatocytes or immune cells, like neutrophils. Interestingly, the strongest RA associated EV proteins were inflammatory molecules, like SAA1 and S100A9, already suggested as biomarkers in RA. Furthermore, the RA associated EV proteins were generally not correlated with total serum protein levels, stressing the importance of EV transport of inflammatory proteins in RA pathogenesis.

• Rydland, Anne; Heinicke, Fatima; Flåm, Siri Tennebø; Mjaavatten, Maria Dahl & Lie, Benedicte Alexandra (2023). Small extracellular vesicles have distinct CD81 and CD9 tetraspanin expression profiles in plasma from rheumatoid arthritis patients. Clinical and Experimental Medicine. ISSN 1591-8890. 23, p. 2867–2875. doi: 10.1007/s10238-023-01024-1. Full text in Research Archive Show summary

  Abstract Extracellular vesicles (EVs) are implicated in the pathogenesis of rheumatoid arthritis (RA) but little is known about the composition of specific small EV (sEV) subpopulations. This study aimed to characterize the CD63, CD81 and CD9 tetraspanin profile in the membrane of single EVs in plasma from treatment naïve RA patients and assess potential discrepancies between methotrexate (MTX) responder groups. EVs isolated from plasma were characterized using transmission electron microscopy, and detection of surface markers (CD63, CD81 and CD9) on single EVs was performed on the ExoView platform. All RA patients ( N  = 8) were newly diagnosed, treatment naïve, females, ACPA positive and former smokers. The controls ( N  = 5) were matched for age and gender. After three months of MTX treatment, responders ( N  = 4) were defined as those with ΔDAS28 > 1.2 and DAS28 ≤ 3.2 post-treatment. The isolated EVs were 50–200 nm in size. The RA patients had a higher proportion of both CD9 and CD81 single positive sEVs compared to healthy controls, while there was a decrease in CD81/CD9 double positive sEVs in patients. Stratification of RA patients into MTX responders and non-responders revealed a distinctly higher proportion of CD81 single positive sEVs in the responder group. The proportion of CD81/CD9 double positive sEVs (anti-CD9 captured) was lower in the non-responders, but increased upon 3 months of MTX treatment. Our exploratory study revealed distinct tetraspanin profiles in RA patients suggesting their implication in RA pathophysiology and MTX treatment response.

• Heinicke, Fatima; Zhong, Xiangfu; Flåm, Siri Tennebø; Breidenbach, Johannes; Leithaug, Magnus & Mæhlen, Marthe Thoresen [Show all 14 contributors for this article] (2021). MicroRNA Expression Differences in Blood-Derived CD19+ B Cells of Methotrexate Treated Rheumatoid Arthritis Patients. Frontiers in Immunology. 12. doi: 10.3389/fimmu.2021.663736. Full text in Research Archive Show summary

  Rheumatoid arthritis (RA) is a complex disease with a wide range of underlying susceptibility factors. Recently, dysregulation of microRNAs (miRNAs) in RA have been reported in several immune cell types from blood. However, B cells have not been studied in detail yet. Given the autoimmune nature of RA with the presence of autoantibodies, CD19+ B cells are a key cell type in RA pathogenesis and alterations in CD19+ B cell subpopulations have been observed in patient blood. Therefore, we aimed to reveal the global miRNA repertoire and to analyze miRNA expression profile differences in homogenous RA patient phenotypes in blood-derived CD19+ B cells. Small RNA sequencing was performed on CD19+ B cells of newly diagnosed untreated RA patients (n=10), successfully methotrexate (MTX) treated RA patients in remission (MTX treated RA patients, n=18) and healthy controls (n=9). The majority of miRNAs was detected across all phenotypes. However, significant expression differences between MTX treated RA patients and controls were observed for 27 miRNAs, while no significant differences were seen between the newly diagnosed patients and controls. Several of the differentially expressed miRNAs were previously found to be dysregulated in RA including miR-223-3p, miR-486-3p and miR-23a-3p. MiRNA target enrichment analysis, using the differentially expressed miRNAs and miRNA-target interactions from miRTarBase as input, revealed enriched target genes known to play important roles in B cell activation, differentiation and B cell receptor signaling, such as STAT3, PRDM1 and PTEN. Interestingly, many of those genes showed a high degree of correlated expression in CD19+ B cells in contrast to other immune cell types. Our results suggest important regulatory functions of miRNAs in blood-derived CD19+ B cells of MTX treated RA patients and motivate for future studies investigating the interactive mechanisms between miRNA and gene targets, as well as the possible predictive power of miRNAs for RA treatment response.

• Heinicke, Fatima; Zhong, Xiangfu; Zucknick, Karola Manuela; Breidenbach, Johannes; Sundaram, Arvind Yegambaram Meenakshi & Flåm, Siri Tennebø [Show all 11 contributors for this article] (2020). An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits. RNA Biology. ISSN 1547-6286. 17(9), p. 1284–1292. doi: 10.1080/15476286.2020.1761081. Full text in Research Archive Show summary

  High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.

• Zhong, Xiangfu; Heinicke, Fatima; Lie, Benedicte Alexandra & Rayner, Simon (2019). Accurate Adapter Information Is Crucial for Reproducibility and Reusability in Small RNA Seq Studies. Non-coding RNA. doi: 10.3390/ncrna5040049. Full text in Research Archive Show summary

  A necessary pre-processing data analysis step is the removal of adapter sequences from the raw reads. While most adapter trimming tools require adapter sequence as an essential input, adapter information is often incomplete or missing. This can impact quantification of features, reproducibility of the study and might even lead to erroneous conclusions. Here, we provide examples to highlight the importance of specifying the adapter sequence by demonstrating the effect of using similar but different adapter sequences and identify additional potential sources of errors in the adapter trimming step. Finally, we propose solutions by which users can ensure their small RNA-seq data is fully annotated with adapter information.

• Heinicke, Fatima; Zhong, Xiangfu; Zucknick, Manuela; Breidenbach, Johannes; Sundaram, Arvind Yegambaram Meenakshi & Flåm, Siri Tennebø [Show all 21 contributors for this article] (2019). Systematic assessment of commercially available low-input miRNA library preparation kits. RNA Biology. ISSN 1547-6286. 17(1), p. 75–86. doi: 10.1080/15476286.2019.1667741. Full text in Research Archive Show summary

  High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

• Zhong, Xiangfu; Heinicke, Fatima & Rayner, Simon (2019). miRBaseMiner, a tool for investigating miRBase content. RNA Biology. ISSN 1547-6286. 16(11), p. 1534–1546. doi: 10.1080/15476286.2019.1637680. Full text in Research Archive Show summary

  microRNAs are small non-coding RNA molecules playing a central role in gene regulation. miRBase is the standard reference source for analysis and interpretation of experimental studies. However, the richness and complexity of the annotation is often underappreciated by users. Moreover, even for experienced users, the size of the resource can make it difficult to explore annotation to determine features such as species coverage, the impact of specific characteristics and changes between successive releases. A further consideration is that each new miRBase release contains entries that have had limited review and which may subsequently be removed in a future release to ensure the quality of annotation. To aid the miRBase user, we developed a software tool, miRBaseMiner, for investigating miRBase annotation and generating custom annotation sets. We apply the tool to characterize each release from v9.2 to v22 to examine how annotation has changed across releases and highlight some of the annotation features that users should keep in mind when using for miRBase for data analysis. These include: (1) entries with identical or very similar sequences; (2) entries with multiple annotated genome locations; (3) hairpin precursor entries with extremely low-estimated minimum free energy; (4) entries possessing reverse complementary; (5) entries with 3ʹ poly(A) ends. As each of these factors can impact the identification of dysregulated features and subsequent clinical or biological conclusions, miRBaseMiner is a valuable resource for any user using miRBase as a reference source.

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• Heinicke, Fatima (2025). Zooming in on CD19+ B Cells in DMARD-naïve RA Patients Using Single-Cell Sequencing. doi: https:/www.tandfonline.com/doi/full/10.1080/03009742.2025.2533698#d1e57. Full text in Research Archive Show summary

  Author: Fatima Heinicke, Maria Dehli Vigeland, Siri Tennebø Flåm, Siri Lillegraven, Nina Paulshus Sundlisæter, Maria Mjaavatten, Silje Syversen, Espen Haavardsholm, Benedicte A. Lie Background: Our knowledge of the role of B cells in rheumatoid arthritis (RA) pathogenesis has expanded beyond autoantibody production like ACPA. They contribute to synovial inflammation and bone destruction through antigen presentation and cytokine release [1,2]. It was further shown that estimated naïve B cell numbers in blood samples increased about two weeks before a disease flare [3]. Clinically, medications that deplete CD20+ B cells are effectively utilized [4]. We now know that B cells perform various functions depending on their developmental stage. Advances in technology, specifically single-cell sequencing, now enable us to study those developmental stages and gene expression for single cells. While the number of individuals included in single-cell analysis remains often limited, the capacity of this technology can be complemented by RNA bulk-sequencing analysis. Although bulk-sequencing lacks single-cell resolution, it can encompass larger cohorts, providing valuable insights. We have previously observed significant differences in the gene expression of blood-derived CD19+ B cells between newly-diagnosed DMARD-naïve RA patients and healthy controls using RNA bulk-sequencing. Aiming to deepen our understanding of their cellular and molecular roles in RA pathogenesis, we conducted single-cell sequencing on a subset of patients in this study. Methods: Patients diagnosed with RA according to the 2010 Rheumatoid Arthritis classification criteria were included in the current study. The patients are part of the Norwegian Very Early Arthritis Clinic (NOR-VEAC) observational study. Age-, gender-, and smoking status-matched controls were sourced from the Norwegian bone marrow registry. After blood withdrawal, CD19+ B cells were isolated using magnetic beads. CD19+B cells from a subset of patients (n=3) and controls (n=3) were further processed for single-cell sequencing. The single-cell data were analyzed using Seurat v5.2.1, Harmony for data integration and miloR v2.2.0 for differential abundance analysis. Results: The newly-diagnosed DMARD-naïve RA patients (n=3) and healthy controls (n=3) included in this single cell study had a median age of 51 years (range: 50-59) and all reported being former smokers, with the exception of one control who still smokes. Two out of three individuals in each group were female. The newly-diagnosed DMARD-naïve RA patients were all ACPA+ and RF+, with a median DAS28 of 5.6 (range: 3.5-6.2) at the time of inclusion. Single cell sequencing of altogether 68,538 cells revealed that the largest proportion in the blood-derived CD19+ B cells were naïve and memory B cells for both patients and controls. The other cell populations were identified as likely-progenitor-, age-associated B- and plasma cells. The previously generated bulk gene expression profiles of CD19+ B cells from a larger cohort of newly-diagnosed DMARD-naïve RA patients and healthy controls, from whom the individuals of the current study were derived, revealed 13 significantly differentially expressed genes (padj<0.1) among them SMIM14, RNF141, ACAA2, H1-4 and POP4. In the present study, we verified those expression differences for all 13 genes using single cell sequencing. Further, single cell sequencing enabled us to investigate whether these genes demonstrate specificity to certain subpopulations of CD19+ B cells. SMIM14 and RNF141 where thereby found to exhibite the highest expression levels in progenitor, naïve B, and memory B cells, while ACAA2, H1-4, and POP4 showed the highest expression in plasma cells. The single-cell dataset enabled us additionally to investigate differences in the abundance of CD19+ B cell subpopulations. Our initial analysis indicated that the majority of subpopulations were uniformly represented between newly-diagnosed DMARD-naïve RA patients and healthy controls. However, we identified an exception within a memory B cell subpopulation characterized by high expression of SOX5, where patients exhibited lower abundances compared to controls. Conclusions: By comparing gene expression between newly-diagnosed DMARD-naïve RA patients and healthy controls, variations in expression profiles may highlight molecular pathways involved in the disease. We could confirm the expression of our 13 differentially expressed genes in an independent blood-derived B cell reference [5]. However, none had previously been linked to RA to the best of our knowledge. SOX5-expressing cells, which we found in lower abundance within a memory B cell subpopulation in RA patients compared to controls, were previously reported to play a role in late B cell development by reducing their proliferation capacity [6]. Furthermore, we plan to expand our study by analyzing (1) the same patients after three months of methotrexate treatment and (2) RA patients in stable remission receiving methotrexate treatment.

• Vigeland, Maria Dehli; Heinicke, Fatima; Flåm, Siri Tennebø; Lillegraven, Siri; Mjaavatten, Maria Dahl & Syversen, Silje Watterdal [Show all 8 contributors for this article] (2025). ABS0248 METHOTREXATE TREATMENT CHANGES GENE EXPRESSION IN CD19+ B-CELLS FROM RHEUMATOID ARTHRITIS PATIENTS. Annals of the Rheumatic Diseases. ISSN 0003-4967. 84, p. 1950–1950. doi: 10.1016/j.ard.2025.06.1500. Full text in Research Archive
• Heinicke, Fatima; Rieder, Anne; Samuelsen, Anne Berit C. & Knutsen, Svein Halvor (2015). Establishing a gene expression system to screen the effects of dietary fibers and their metabolites on selected aspects of colon cancer development. Universitetet i Oslo. Full text in Research Archive

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